Part:BBa_K538000:Experience

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Applications of BBa_K538000

This brick's developers, team [http://2011.igem.org/Team:Amsterdam Amsterdam 2011], partially characterised this brick's effect on E. coli 's cold resistance via BBa_K538200. Literature only reports Cpn10 being functional in presence of its partner protein Cpn60, so in order to fully characterise it, it must be coexpressed with Cpn60. At the time of this writing, this is unfeasible, because attempts to assemble a Cpn60 generator are thusfar unsuccesful. However, following the methods described on their [http://2011.igem.org/Team:Amsterdam/Labwork/Characterisation characterisation] page, they did at least verify the hypothesis Cpn10 does not, on its own, significantly influence growth rate at suboptimal temperatures.

In addition to investigating Cpn10 's effect on growth at suboptimal temperatures, cells expressing Cpn10 also had their freeze/thaw cycle survival rate compared against that of an E. coli strain containing an empty vector. Much to the team's surprise, the preliminary data suggests Cpn10 expression can, even in absence of Cpn60, affect the cold resistance of E. coli ! While it's not as effective as CspC, three cultures expressing Cpn10 still contained 15 to 27 viable cells per 100µL after 3 freeze/thaw cycles, whereas three control cultures, containing an empty vector, barely showed any CFUs on plate at all after the third cycle. A single colony was observed on the three control plates. See also figure 1, below.

Figure 1. Plates of three different E. coli strains before (first column, diluted 10.000x) and after (second, third and fourth column, undiluted) three freeze/thaw cycles. The top row of plates were loaded with E. coli comprising an empty vector. The bacteria on the plates in the middle row expressed CspC, and those in the bottom row expressed Cpn10. The bacteria comprising CspC were about 4 times as numerous as the other strains from the start, but after 3 freeze/thaw cycles, their survival rate relative to the amount of CFUs before the first cycle was higher as well. On average, an estimated 0.121% of CspC 's initial cell count remained viable, whereas 0.01% of the Cpn10 expressing cells survived. Practically none of the cells with an empty vector survived a third freeze/thaw cycle.

If the observation Cpn10 influences E. coli 's ability to survive freeze/thaw cycles in absence of Cpn60 can be reproduced, this constitutes an interesting discovery. Cpn60 has already been reported to function in absence of Cpn10.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1138217/pdf/biochemj00085-0050.pdf] However, to the best of our knowledge, the reverse - Cpn10 functioning in absence of Cpn60 - has never been reported. Based on structural analysis, it has been suggested the Cpn10 of Mycobacterium tuberculosis can bind substrates in absence of Cpn60,[http://jb.asm.org/cgi/reprint/185/14/4172.pdf] but demonstrating the Cpn10 of Oleispira antarctica can enhance the cold resistance of E. coli in absence of Cpn60 would be a steady step towards validating this otherwise unconfirmed suggestion.

1. Staniforth, Burston, Atkinson & Clark Affinity of chaperonin-60 for a protein substrate and its modulation by nucleotides and chaperonin-10. Biochem J. 300 (3), 651-658 (1994)
2. Robberts et al. Mycobacterium tuberculosis chaperonin 10 heptamers self-associate through their biologically active loops. J. Bacteriol. 185, 4172-4185 (2003)

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